This project studies physiological and cellular aspects of neuronal calcium signaling, with long-range emphasis on postsynaptic responses in large central nervous system neurons. Neurons respond to synaptic stimuli with a rise in cytosolic free Ca2+ concentration ([Ca2+]i) that is strongly modulated by the activity of intracellular Ca stores. This activity plays an important role in spatio-temporally shaping cytosolic Ca2+ signals. We had earlier shown that in frog sympathetic neurons ? an excellent model for studying intracellular Ca2+ dynamics ? increases in [Ca2+]i are accompanied by large, reversible elevations in total mitochondrial calcium concentration ([Ca]m) that give rise to steep spatial gradients in the intracellular distribution of Ca. We have now characterized the function of the second major Ca2+-regulating organelle, the endoplasmic reticulum (ER), whose role is generally thought to be amplification of Ca2+ signals by triggered Ca2+ release from its internal store, a process known as "calcium-induced calcium release" (CICR). We find, however, that the behavior of the ER is more complex. At low levels of Ca2+ entry (and therefore low [Ca2+]i) the ER actually accumulates Ca, thus acting as a Ca2+ buffer. However, these neurons exhibit a progressive transition from Ca2+ buffering to triggered Ca2+ release as [Ca2+]i increases to approx. 1uM. In addition, it was found that Ca2+ release is first triggered in peripheral ER cisternae, so that both Ca2+ uptake and release can occur simultaneously different regions of the same cell. Finally, the spatial gradient of ER Ca2+ transport is reciprocal to that of mitochondrial Ca2+ uptake, suggesting cooperation between these organelles. Functional interdependence between mitochondria and ER is supported by data showing that inhibition of ER Ca2+ uptake leads to compensatory Ca sequestration by mitochondria. Reciprocally, suppression of mitochondrial Ca accumulation under equivalent conditions results in increased ER Ca2+ uptake. In a variety of neurons, including hippocampal pyramidal cells, large increases in [Ca2+]i activate several key kinases, whereas lower [Ca2+]i enhances phosphatase activity. This Ca2+-dependent rebalancing of the phosphorylation status of certain key enzymes, e.g., Ca/calmodulin-dependent kinases (CaMKs), appears to control the activity of pathways central to neuronal plasticity, examples being synaptically-evoked gene expression and LTP induction. We previously found that mitochondrial Ca accumulation, occurring mainly in peripheral locations during Ca2+ entry, leads to an increase in the production of superoxide radicals (O2-). As superoxide is known to activate protein kinases and, complementarily, to inhibit phosphatases, we examined the relationship between [Ca2+]i, O2-, and phosphorylation of CaMKII and protein kinase C (PKC), with the goal of elucidating the molecular mechanisms underlying this signaling pathway. We find that mitochondrial Ca2+ uptake that augments O2- production is dependent on stimulus strength and duration. Measurements obtained with fluorescent probes in living hippocampal neurons demonstrate that weak stimuli (5Hz/18s or longer) elicit low, sustained [Ca2+]i plateaus, but do not induce O2- generation, while stronger stimuli (50 or 100Hz/18s, and 90 mM K+) induce large Ca2+ spikes and increased mitochondrial O2- production. Using quantitative immunocytochemistry, we have further evaluated the role of mitochondria in linking cytosolic Ca2+ entry to the fine-tuning of cellular phosphorylation. One major pathway appears to target the phosphorylation of CaMKs. By inhibiting an array of phosphatases (PP1, PP2A and/or PP2B), mitochondrial O2- enhances autophosphorylation of CaMKII (important for LTP induction), as well as CaMKIV-dependent phosphorylation of CREB (important for gene expression). In contrast, mitochondrial O2- appears to modulate PKC by a mechanism that does not involve phosphatase activity. Excessive mitochondrial Ca accumulation is thought to play a crucial role in excitotoxicity. We have found that exposure of hippocampal neurons to glutamate or NMDA results in dramatic changes in the ionic content of these cells, specifically, an elevation of free and total cytosolic Ca, as well as a gain in Na and a loss of K. Treated neurons also exhibited extremely high levels of intramitochondrial Ca. These changes were largely reversible, in that normal cytosolic ion levels were restored after agonist removal, even though certain mitochondria maintained elevated Ca for prolonged periods. Inhibition of mitochondrial Ca2+ uptake by FCCP appeared to be neuroprotective, despite the fact that FCCP increased the amplitude of cytosolic Ca2+ transients. The results suggest that elevation of [Ca2+]i alone, without elevation of mitochondrial Ca, is not sufficient to induce cell death, and leads to the hypothesis that cell fate may depend on the level of Ca within mitochondria.